Increased frequency of multiple sclerosis among patients with bullous pemphigoid: a population-based cohort study on comorbidities anchored around the diagnosis of bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is a disease of the elderly and may be associated with neurological and cardiovascular diseases and diabetes. Mortality rates strongly exceed those of the background population. OBJECTIVES: To investigate the frequency of comorbidities and their temporal relation to BP. METHODS: A register-based matched-cohort study on all Danish patients with a hospital-based diagnosis of BP (n = 3281). The main outcomes were multiple sclerosis (MS), Parkinson disease (PD), Alzheimer disease (AD), stroke, diabetes types 1 and 2, malignancies, ischaemic heart disease (IHD), hypertension and eventually death. RESULTS: At baseline, patients with BP had increased prevalences of MS [odds ratio (OR) 9·7, 95% confidence interval (CI) 6·0-15·6], PD (OR 4·2, 95% CI 3·1-5·8), AD (OR 2·6, 95% CI 1·8-3·5) and stroke (OR 2·7, 95% CI 2·4-2·9). Furthermore, malignancies, cardiovascular disease and diabetes were over-represented among patients with BP: type 1 diabetes (OR 3·1, 95% CI 2·5-3·8), type 2 diabetes (OR 2·3, 95% CI 2·0-2·6), malignancies (OR 1·3, 95% CI 1·1-1·4), IHD (OR 1·7, 95% CI 1·5-1·9) and hypertension (OR 2·0, 95% CI 1·8-2·2). During follow-up, the risk of MS was significantly higher among patients with BP [hazard ratio (HR) 9·4, 95% CI 4·9-18·0], even if events during the first year after diagnosis of BP were excluded (HR 5·1, 95% CI 2·3-11·3). Patients with BP had an average increased mortality rate of 2·04 (95% CI 1·96-2·13). CONCLUSIONS: We discovered a significantly increased frequency of MS among patients with BP. At the time of diagnosis, patients with BP had an excessive number of comorbidities and an increased mortality rate over the following years.

Cancer incidence among patients with a hospital diagnosis of pruritus: a nationwide Danish cohort study.

BACKGROUND: Pruritus is a frequent complaint in patients with cancer. However, no large study has examined pruritus as a marker of undiagnosed cancer. OBJECTIVES: To examine the association between inpatient, outpatient and emergency hospital diagnoses of pruritus and subsequent cancer diagnoses. METHODS: In this nationwide Danish cohort study, we used medical databases to identify all patients (n = 12,813) with a diagnosis of pruritus during the period 1978-2011 and followed them until a first-time cancer diagnosis, emigration, death or 31 December 2011. We computed standardized incidence ratios (SIRs) for cancer as the observed to expected number of cancers based on national cancer incidence rates. We calculated the 1-year absolute risk of cancer, treating death as a competing risk. RESULTS: The overall SIR of cancer was 1.13 [95% confidence interval (CI) 1.07-1.20]: 1.22 (95% CI 1.13-1.33) among men and 1.05 (95% CI 0.97-1.14) among women. The SIR was 1.20 (95% CI 1.08-1.33) among patients with a previous diagnosis of dermatological disease and 1.10 (95% CI 1.02-1.18) among patients without such a diagnosis. Both haematological and various solid cancers were observed at increased rates. Overall, the highest SIRs were observed during the first 3 months of follow-up, declining rapidly thereafter. The 1-year absolute risk of a cancer diagnosis was 1.63% and 155 patients with pruritus would have needed to be examined to detect one excess cancer. CONCLUSIONS: Pruritus may be a marker of occult cancer. Further studies are needed to assess the prognostic benefit of screening for cancer in patients with pruritus.

Use of oral bisphosphonates and risk of venous thromboembolism: a population-based case-control study.

UNLABELLED: Oral bisphosphonates are widely used for fracture prevention, but there is a concern regarding potential adverse cardiovascular effects of bisphosphonates. In this large, population-based case-control study, we found no evidence of an association between bisphosphonate use and risk of venous thromboembolism (VTE). INTRODUCTION: We examined the relation between the use of oral bisphosphonates for osteoporosis and the risk of VTE. METHODS: We conducted a population-based case-control study in Northern Denmark (population, 1.7 million). Using the Danish National Registry of Patients, we identified all women with a first-time hospital diagnosis of VTE between 1999 and 2006. For each case, we selected up to ten female population controls, matched on date of the index VTE event and age. Data on use of oral bisphosphonates, other medications, and comorbidity were obtained from medical databases. We used logistic regression to estimate odds ratios (OR) for VTE associated with bisphosphonate users while adjusting for potential confounding factors. RESULTS: Four thousand one hundred ninety-three cases and 41,197 controls were included in the study. One hundred forty-nine cases (3.6%) and 1,078 controls (2.6%) were current bisphosphonate users. The adjusted OR for VTE among the current bisphosphonate users compared with nonusers was 1.03 (95% confidence interval (CI): 0.84-1.26), and when restricted to cases of unprovoked thromboembolism, the adjusted OR was 1.08 (95% CI: 0.82-1.42). There was no association either for pulmonary embolism or for deep venous thrombosis. CONCLUSION: We found no evidence of an association of oral bisphosphonate use with the risk of VTE.

Specific inhibition of eIF-5A and collagen hydroxylation by a single agent. Antiproliferative and fibrosuppressive effects on smooth muscle cells from human coronary arteries.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.

Lifetime success and interactions of farm salmon invading a native population.

Farm Atlantic salmon escape and invade rivers throughout the North Atlantic annually, which has generated growing concern about their impacts on native salmon populations. A large-scale experiment was therefore undertaken in order to quantify the lifetime success and interactions of farm salmon invading a Norwegian river. Sexually mature farm and native salmon were genetically screened, radio tagged and released into the River Imsa where no other salmon had been allowed to ascend. The farm fishes were competitively and reproductively inferior, achieving less than one-third the breeding success of the native fishes. Moreover, this inferiority was sex biased, being more pronounced in farm males than females, resulting in the principal route of gene flow involving native males mating with farm females. There were also indications of selection against farm genotypes during early survival but not thereafter. However, evidence of resource competition and competitive displacement existed as the productivity of the native population was depressed by more than 30%. Ultimately, the lifetime reproductive success (adult to adult) of the farm fishes was 16% that of the native salmon. Our results indicate that such annual invasions have the potential for impacting on population productivity, disrupting local adaptations and reducing the genetic diversity of wild salmon populations.

[Emergency care during ambulatory vasectomy]. / Beredskab ved ambulant vasektomi.

We describe a case of cardiac arrest during outpatient vasectomy. The possible mechanisms and need for monitoring are discussed. We recommend that the surgeon should be able to provide basic life support during outpatient vasectomy. Routine electronic monitoring during and after the procedure is probably not warranted and should at least await clinical studies on cardiac function and morbidity during and after the procedure.

Kidney disease and risk of venous thromboembolism: a nationwide population-based case-control study.

BACKGROUND: Chronic kidney disease is associated with hemostatic derangements, including both procoagulant activity and platelet dysfunction, which may influence the risk of venous thromboembolism. However, data associating kidney disease with risk of venous thromboembolism are sparse. OBJECTIVES: We examined whether kidney disease is associated with increased risk of venous thromboembolism. METHODS: We conducted this nationwide case-control study using data from medical databases. We included 128,096 patients with a hospital diagnosis of VTE in Denmark between 1980 and 2010 (54,473 had pulmonary embolism and 73,623 had deep venous thrombosis only) and 642,426 age- and gender-matched population controls based on risk-set sampling. We identified all previous hospital diagnoses of kidney disease, including nephrotic syndrome, glomerulonephritis without nephrotic syndrome, hypertensive nephropathy, chronic pyelonephritis/interstitial nephritis, polycystic kidney disease, diabetic nephropathy, or other kidney diseases. We used conditional logistic regression models to compute odds ratios (ORs) for venous thromboembolism with adjustment for potential confounders. RESULTS: Kidney disease was associated with an adjusted OR for venous thromboembolism ranging from 1.41 (95% CI, 1.22-1.63) for hypertensive nephropathy to 2.89 (95% CI, 2.26-3.69) for patients with nephrotic syndrome. The association was strongest within the first 3 months after a diagnosis of chronic kidney disease (adjusted OR for nephrotic syndrome = 23.23; 95% CI, 8.58-62.89), gradually declining thereafter. The risk, however, remained elevated for more than 5 years, especially in patients with nephrotic syndrome and glomerulonephritis. CONCLUSIONS: Kidney diseases, in particular nephrotic syndrome and glomerulonephritis, were associated with an increased risk of venous thromboembolism.

Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer in which the alpha subunits contribute to most parts of the two catalytic sites. To study the roles of histidine and cysteine residues in this catalytic activity we converted all 5 histidines that are conserved between species, 4 nonconserved histidines, and 3 conserved cysteines of the human alpha subunit individually to serine and expressed the mutant alpha subunits together with the wild-type beta subunit in insect cells by means of baculovirus vectors. Mutation of any of the 3 conserved histidines, residues 412, 483, and 501, inactivated the enzyme completely or essentially completely, with no effect on tetramer assembly or binding of the tetramer to poly(L-proline). These histidines are likely to provide the three ligands needed for the binding of Fe2+ to a catalytic site. Mutation of either of the other 2 conserved histidines reduced the amount of enzyme tetramer by 20-25% and the activity of the tetramer by 30-60%. Mutation of the nonconserved histidine 324 totally prevented tetramer assembly, whereas mutation of the 3 other nonconserved histidines had no effects. Two of the 3 cysteine to serine mutations, those involving residues 486 and 511, totally prevented tetramer assembly under the present conditions, whereas the third, involving residue 150, had only a minor effect in reducing tetramer assembly and activity. The data do not support previous suggestions that cysteine residues are involved in Fe2+ binding sites. Additional mutagenesis experiments demonstrated that the two glycosylated asparagines have no role in tetramer assembly or catalytic activity.

Expression of wild-type and modified proalpha chains of human type I procollagen in insect cells leads to the formation of stable [alpha1(I)]2alpha2(I) collagen heterotrimers and [alpha1(I)]3 homotrimers but not [alpha2(I)]3 homotrimers.

Insect cells coinfected with a baculovirus coding for the proalpha1(I) chain of human type I procollagen and a double promoter virus coding for the alpha and beta subunits of human prolyl 4-hydroxylase produced homotrimeric [proalpha1(I)]3 procollagen molecules. The use of an additional virus coding for the proalpha2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proalpha1 to proalpha2 chains of type I procollagen (proalpha1(I) and proalpha2(I) chains, respectively), unless the proalpha1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proalpha1(I) chain with those of the proalpha1(III) chain increased level of expression of the proalpha1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proalpha2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. The Tm of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42-43 degrees C when determined by circular dichroism. The wild-type proalpha2(I) chain formed no homotrimers. Replacement of the C propeptide of the proalpha2(I) chain with that of the proalpha1(I) chain or proalpha1 chain of type III procollagen (proalpha1(III) chain) led to the formation of homotrimers, but the alpha2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 degrees C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proalpha2(I)]3 molecules in insect cells.

Type XIII collagen forms homotrimers with three triple helical collagenous domains and its association into disulfide-bonded trimers is enhanced by prolyl 4-hydroxylase.

Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-terminal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen alpha chains were found to associate into disulfide-bonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen alpha chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities (T(m)) of the COL1, COL2, and COL3 domains were 38, 49 and 40 degrees C, respectively. The T(m) of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.

Characterization of human type III collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit.

An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-alpha 1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the Tm of the triple helices was only about 32-34 degrees C. Coexpression of the pro-alpha1(III) chains with the alpha and beta subunits of human prolyl 4-hydroxylase increased the Tm to about 40 degrees C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-alpha 1(III) chains and alpha1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 microg of cellular type III collagen (120 microg of procollagen) per 5 X 10(6) cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and Tm of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.

The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity.

The microsomal triacylglycerol transfer protein (MTP), an alpha beta dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The beta subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the alpha subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the alpha and beta subunits were co-expressed, soluble alpha beta dimers were formed with significant triacylglycerol transfer activity. Expression of the alpha subunit with a mutant protein disulphide isomerase polypeptide in which both -CGHC- catalytic sites had been inactivated also yielded alpha beta dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the beta subunit in MTP seems to be to keep the alpha subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the beta subunit is not required for this function.